The detoxification of ammonia in humans occurs primarily in the liver through the combined action of five enzymes. These enzymes catalyze the transformation of ammonia into urea for eventual disposition via the urine. The primary objective of this proposal is to elucidate the detailed mechanisms of action and regulatory properties of carbamyl phosphate synthetase (CPS) and argininosuccinate synthetase. The specific aims with the CPS enzyme are as follows: (1) Carbamyl phosphate synthetase activity is regulated by both positive and negative allosteric effectors. A thermodynamic model for this regulatory mechanism will be constructed using a steady-state and rapid reaction kinetic analysis of the effect of these regulatory molecules on the activity of carbamyl phosphate synthetase. (2) The enzyme from E. coli is composed of a large subunit (MW approximately 120,000) and a small subunit (MW approximately 40,000). The primary amino acid sequence of the large subunit has revealed a high degree of homology between the amino-terminal and carboxyl-terminal halves. The function of each of these halves (domains?) will be probed by covalent labelling of the catalytic and regulatory sites with reactive analogs of the substrate and nucleotide effectors. Mutant enzymes will be constructed by genetic and chemical means as a method for determining whether these domains have independent functions. (3) The function of amino acids in the active sit of CPS will be analyzed by pH profile and inactivation studies with group specific reagents. With the enzyme argininosuccinate synthetase the specific aims are to establish the structures of the intermediates formed, at the enzyme active site and to elucidate the function of the amino acids at the active site in catalysis and binding of substrates and products. The identity of the reactive intermediates will be made by the, synthesis of chemical analogs and direct observation by NMR spectroscopy. The identity and function of amino acid residues at the active site will be probed by pH-activity analysis and the effects of group-specific reagents.